EP0421736A2
Expired / Lapsed
EP
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Method and apparatus for measuring optical properties of biological speciments
Assignee: Individual
•
Filing: 1990-10-02
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Granted: —
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Expires: 2010-10-02
Abstract
Abstract The invention relates to an apparatus and methods for generating multiparameter optical data that characterize a population of cells. The invention includes the steps of scanning the cell population with a beam to produce sets of digital data samples, each sample set of digital data representing multiparameter optical interactions from a specific location within the cell population; storing the digital data, e.g., in a computer memory; locating a cell within the population, e.g., by comparing the digital data to a preselected threshold value; defining a neighborhood around the digital data representing the located cell; estimating a background level for the neighborhood based upon digital data corresponding to locations outside the neighborhood; and correcting each of the samples corresponding to the neighborhood with the estimated neighborhood background level to generate the optical data. The invention further relates to specific methods of background correction and data calibration as well as specific sampling features to enable precise estimates of multiple cellular constituents or other cell properties at high rates of speed.
Inventors
- 👤 Louis A. Kamentsky
- 👤 Lee D. Kamentsky
Bibliographic Details
Application Number
—
Jurisdiction
EP
Data Source
google_patents
Last Synchronized
2026-10-26 22:20
CPC Classifications
G; G01; G01N; G01N15/00; G01N15/10; G01N15/14; G01N15/1468; G01N15/147; G06; G06V; G06V20/00; G06V20/60; G06V20/69
Claims Analysis
Total Claims: 56
Claim 1
Independent Claim
103 words
A method for generating optical data that characterizes a population of cells, the method comprising: (a) scanning the cell population with a beam to produce digital data samples, different said samples representing different locations within the cell population; (b) storing said digital data; (c) locating a cell within said population; (d) defining a neighborhood around the located cell; (e) estimating a background level for the neighborhood based upon stored digital data corresponding to locations outside said neighborhood; and (f) correcting each of said digital data samples corresponding to the neighborhood with the estimated neighborhood background level to generate the optical data. said sample
Claim 2
Independent Claim
103 words
A method for generating optical data that characterizes a population of cells, the method comprising: (a) scanning the cell population with a beam to produce digital data samples, different said samples representing different locations within the cell population; (b) storing said digital data; (c) locating a cell within said population; (d) defining a neighborhood around the located cell; (e) estimating a background level for the neighborhood based upon stored digital data corresponding to locations outside said neighborhood; and (f) correcting each of said digital data samples corresponding to the neighborhood with the estimated neighborhood background level to generate the optical data. said sample
Claim 3
Dependent Claim
27 words
The method of claim 1, wherein said sample correcting step comprises subtracting the corresponding background level from each of said stored digital data samples in a neighborhood.
Claim 4
Dependent Claim
22 words
The method of claim 1, wherein said digital data samples represent a plurality of morphological properties of each cell in the population.
Claim 5
Dependent Claim
15 words
The method of claim 1, wherein said neighborhood comprises a given number of sample points.
Claim 6
Dependent Claim
27 words
The method of claim 4, wherein said background level for a neighborhood comprises a plurality of individual background levels, one for each sample point in said neighborhood.
Claim 7
Dependent Claim
43 words
The method of claim 1 wherein said cell population is on a movable surface, said scanning proceeds in synchrony with a stepping motion of said surface and digital location data is generated from which the location corresponding to a sample can be derived.
Claim 8
Dependent Claim
16 words
The method of claim 6, wherein said cells on said movable surface can be visually observed.
Claim 9
Dependent Claim
35 words
The method of claim 1, wherein said neighborhood background level is determined by finding minimum digital data sample values in memory locations corresponding to actual locations adjacent the neighborhood for each sample in said neighborhood.
Claim 10
Dependent Claim
26 words
The method of claim 1, wherein said neighborhood background levels are determined by averaging a plurality of digital data sample values in memory outside said neighborhood.
Claim 11
Dependent Claim
75 words
The method of claim 1, wherein said cell population is on a movable surface, said scanning proceeds in synchrony with a stepping motion of said surface, the time corresponding approximately to the time the digital data was acquired is recorded and stored as synchronous digital time data, and digital location data is generated from which the location corresponding to a sample can be derived, whereby each sample has a corresponding digital time point and location.
Claim 12
Independent Claim
44 words
The method of olaim 10, further oomprising the step of rescanning the population of cells, generating new digital data, location data and time data, and comparing said new data with the corresponding data from a previous scan to determine changes in said cell population.
Claim 13
Independent Claim
44 words
The method of olaim 10, further oomprising the step of rescanning the population of cells, generating new digital data, location data and time data, and comparing said new data with the corresponding data from a previous scan to determine changes in said cell population.
Claim 14
Dependent Claim
19 words
The method of claim 1, wherein the size of said neighborhood is set to statistically include a single cell.
Claim 15
Dependent Claim
27 words
The method of claim 1, wherein said locating step comprises detecting a digital data sample corresponding to the edge of the cell first contacted by said beam.
Claim 16
Independent Claim
99 words
A method for generating optical data that characterizes a population of cells, the method comprising: (a) scanning the cell population with a beam with a spot size comparable in size to the cells to be scanned to produce digital data samples, different said samples representing different locations within the cell population and said samples being taken at a sampling rate set such that the distance travelled per time as the spot is scanned between consecutive sampled locations approximates said spot size; and (b) processing the digital data to generate the optical data that characterizes the cells in the population
Claim 17
Independent Claim
99 words
A method for generating optical data that characterizes a population of cells, the method comprising: (a) scanning the cell population with a beam with a spot size comparable in size to the cells to be scanned to produce digital data samples, different said samples representing different locations within the cell population and said samples being taken at a sampling rate set such that the distance travelled per time as the spot is scanned between consecutive sampled locations approximates said spot size; and (b) processing the digital data to generate the optical data that characterizes the cells in the population
Claim 18
Dependent Claim
14 words
The method of claim 14, wherein said spot size is 3 to 50 microns.
Claim 19
Dependent Claim
24 words
The method of claim 14, wherein said processing step comprises correcting said digital data samples with a background level to generate said optical data.
Claim 20
Independent Claim
137 words
A method for generating calibrated optical data that characterizes a population of cells, the method comprising: (a) scanning the cell population on a surface with a beam to produce digital data samples, said beam having a given illumination intensity, different said samples representing different locations within the cell population; (b) storing said digital data; (c) scanning a region of said surface without cells to generate intensity calibration data; (d) generating an intensity normalization factor from said intensity calibration data and storing said intensity normalization factor in memory; (e) locating a cell within said population; (f) defining a neighborhood around the located cell; said neighborhood containing optical data derived from said stored digital data samples within said neighborhood; and (g) calibrating the optical data in said neighborhood with said intensity normalization factor to generate the calibrated optical data.
Claim 21
Independent Claim
137 words
A method for generating calibrated optical data that characterizes a population of cells, the method comprising: (a) scanning the cell population on a surface with a beam to produce digital data samples, said beam having a given illumination intensity, different said samples representing different locations within the cell population; (b) storing said digital data; (c) scanning a region of said surface without cells to generate intensity calibration data; (d) generating an intensity normalization factor from said intensity calibration data and storing said intensity normalization factor in memory; (e) locating a cell within said population; (f) defining a neighborhood around the located cell; said neighborhood containing optical data derived from said stored digital data samples within said neighborhood; and (g) calibrating the optical data in said neighborhood with said intensity normalization factor to generate the calibrated optical data.
Claim 22
Dependent Claim
21 words
The method of claim 17, wherein said optical data calibrating step comprises multiplying said optical data by said intensity normalization factor.
Claim 23
Dependent Claim
20 words
The method of claim 17, wherein said surface without cells is a calibration slide comprising a uniform fluorescent dye surface.
Claim 24
Dependent Claim
19 words
The method of claim 17, wherein said surface without cells is a calibration slide comprising a uniform scattering surface.
Claim 25
Dependent Claim
61 words
The method of claim 17, wherein the derivation of said optical data contained in said neighborhood comprises the steps of: estimating background levels for the individual neighborhoods based upon stored digital data corresponding to locations outside said neighborhood; and correcting each of said digital data samples in each neighborhood with the corresponding estimated neighborhood background level to generate said optical data.
Claim 26
Independent Claim
149 words
A method of measuring cellular properties comprising the steps of: (a) scanning the cell population with a beam to produce digital data samples, different said samples representing different locations within the cell population; (b) storing said digital data; (c) locating a cell within said population; (d) defining a neighborhood around the located cell, said neighborhood containing optical data derived from said stored digital data samples within said neighborhood; (e) summing all samples of optical data in a 14 neighborhood to generate an optical neighborhood value for that neighborhood; (f) comparing each sample in a neighborhood with a preselected morphology threshold to generate a binary neighborhood pattern of samples either above or below said threshold; (g) storing said optical neighborhood value and binary neighborhood pattern in memory; and (h) generating optical properties corresponding to said optical neighborhood value and morphological properties corresponding to said binary neighborhood pattern for each cell.
Claim 27
Independent Claim
149 words
A method of measuring cellular properties comprising the steps of: (a) scanning the cell population with a beam to produce digital data samples, different said samples representing different locations within the cell population; (b) storing said digital data; (c) locating a cell within said population; (d) defining a neighborhood around the located cell, said neighborhood containing optical data derived from said stored digital data samples within said neighborhood; (e) summing all samples of optical data in a 14 neighborhood to generate an optical neighborhood value for that neighborhood; (f) comparing each sample in a neighborhood with a preselected morphology threshold to generate a binary neighborhood pattern of samples either above or below said threshold; (g) storing said optical neighborhood value and binary neighborhood pattern in memory; and (h) generating optical properties corresponding to said optical neighborhood value and morphological properties corresponding to said binary neighborhood pattern for each cell.
Claim 28
Dependent Claim
35 words
The method of claim 22, wherein the generation of said optical properties comprises: applying math functions to one or more said optical neighborhood values to generate optical properties of the cell corresponding to said neighborhood.
Claim 29
Dependent Claim
35 words
The method of claim 22, wherein the generation of said morphological properties comprises: applying boolean functions to one or more said binary neighborhood patterns to generate morphological properties of the cell corresponding to said neighborhood.
Claim 30
Dependent Claim
58 words
The method of claim 22, wherein the derivation of said optical data contained in said neighborhood comprises: estimating a background level for the neighborhood based upon stored digital data corresponding to locations outside said neighborhood; and correcting each of said digital data samples corresponding to the neighborhood with the estimated neighborhood background level to generate said optical data.
Claim 31
Independent Claim
31 words
The method of any one of claims 1, 14, 17, or 22, wherein said digital data samples produced by the cells 3 and the beam are derived from analog optical data.
Claim 32
Dependent Claim
21 words
The method of claim 26, wherein said optical data is fluorescence, forward angle light scatter, extinction, or wide angle light scatter.
Claim 33
Independent Claim
209 words
An apparatus for measuring cellular properties of a population of cells comprising: a beam source for generating a beam of electromagnetic radiation to illuminate the cells to be measured with a beam spot comparable in size to the cells to be scanned, an optical signal being generated as a result of said spot illuminating said cells; a surface upon which the cells are located; an optical path for directing said spot from said beam source to the cells on said surface; a scanner interposed in said optical path between said beam source and the cells for scanning said spot across said surface so that said spot passes over a plurality of cells on said surface; a sensor for measuring said optical signal; an analog-to-digital converter arranged to sample said optical signal at a specified rate and to produce digital data; whereby said sampling rate is set such that the distance traveled per time as the spot is scanned between consecutive sampled locations approximates said spot size; a data processor for deriving optical data from said digital data, generating optical property values from said optical data corresponding to individual cells, and processing said values to determine cellular properties; and a memory for storing said digital data produced by said converter.
Claim 34
Independent Claim
209 words
An apparatus for measuring cellular properties of a population of cells comprising: a beam source for generating a beam of electromagnetic radiation to illuminate the cells to be measured with a beam spot comparable in size to the cells to be scanned, an optical signal being generated as a result of said spot illuminating said cells; a surface upon which the cells are located; an optical path for directing said spot from said beam source to the cells on said surface; a scanner interposed in said optical path between said beam source and the cells for scanning said spot across said surface so that said spot passes over a plurality of cells on said surface; a sensor for measuring said optical signal; an analog-to-digital converter arranged to sample said optical signal at a specified rate and to produce digital data; whereby said sampling rate is set such that the distance traveled per time as the spot is scanned between consecutive sampled locations approximates said spot size; a data processor for deriving optical data from said digital data, generating optical property values from said optical data corresponding to individual cells, and processing said values to determine cellular properties; and a memory for storing said digital data produced by said converter.
Claim 35
Dependent Claim
14 words
The apparatus of claim 28, wherein said spot size is 3 to 50 microns.
Claim 36
Dependent Claim
13 words
The apparatus of claim 28, wherein said scanner is a resonant galvanometer scanner.
Claim 37
Dependent Claim
18 words
The apparatus of claim 28, wherein the wavelength distribution of said beam is controlled by the data processor.
Claim 38
Independent Claim
120 words
An apparatus for generating optical data that characterizes a population of cells comprising: (a) a scanner for scanning the cell population with a beam to produce digital data samples, different said samples representing different locations within the cell population; (b) a memory to store said digital data; (c) means for locating digital data samples in memory corresponding to a cell within said population; (d) means for defining a neighborhood around the located cell; (e) means for estimating a background level for the neighborhood based upon stored digital data corresponding to locations outside said neighborhood; and (f) means for correcting each of said digital data samples corresponding to the neighborhood with the estimated neighborhood background level to generate the optical data.
Claim 39
Independent Claim
120 words
An apparatus for generating optical data that characterizes a population of cells comprising: (a) a scanner for scanning the cell population with a beam to produce digital data samples, different said samples representing different locations within the cell population; (b) a memory to store said digital data; (c) means for locating digital data samples in memory corresponding to a cell within said population; (d) means for defining a neighborhood around the located cell; (e) means for estimating a background level for the neighborhood based upon stored digital data corresponding to locations outside said neighborhood; and (f) means for correcting each of said digital data samples corresponding to the neighborhood with the estimated neighborhood background level to generate the optical data.
Claim 40
Independent Claim
149 words
An apparatus for generating calibrated optical data that characterizes a population of cells comprising: (a) a scanner for scanning the cell population on a surface with a beam to produce digital data samples, said beam having a given illumination intensity, different said samples representing different locations within the cell population, and for scanning a region of said surface without cells to generate intensity calibration data; (b) a memory for storing said digital data; (c) means for generating an intensity normalization factor from said intensity calibration data; (d) means for locating digital data samples in memory corresponding to a cell within said population; (e) means for defining a neighborhood around the located cell; said neighborhood containing optical data derived from said stored digital data samples within said neighborhood; and (g) means for correcting the optical data in said neighborhood with said intensity normalization factor to generate the calibrated optical data.
Claim 41
Independent Claim
149 words
An apparatus for generating calibrated optical data that characterizes a population of cells comprising: (a) a scanner for scanning the cell population on a surface with a beam to produce digital data samples, said beam having a given illumination intensity, different said samples representing different locations within the cell population, and for scanning a region of said surface without cells to generate intensity calibration data; (b) a memory for storing said digital data; (c) means for generating an intensity normalization factor from said intensity calibration data; (d) means for locating digital data samples in memory corresponding to a cell within said population; (e) means for defining a neighborhood around the located cell; said neighborhood containing optical data derived from said stored digital data samples within said neighborhood; and (g) means for correcting the optical data in said neighborhood with said intensity normalization factor to generate the calibrated optical data.
Claim 42
Independent Claim
143 words
An apparatus for measuring cellular properties comprising: (a) a scanner for scanning the cell population with a beam to produce digital data samples, different said samples representing different locations within the cell population; (b) a memory for storing said digital data; (c) means for locating digital data samples in memory corresponding to a cell within said population; (d) means for defining a neighborhood around the located cell; (e) an adder for summing all samples of optical data in a neighborhood to generate an optical neighborhood value for that neighborhood; (f) a comparator for comparing each sample in a neighborhood with a preselected morphology threshold to generate a binary neighborhood pattern of samples either above or below said threshold; and (g) means for generating optical properties corresponding to said optical neighborhood value and morphological properties corresponding to said binary neighborhood pattern for each cell.
Claim 43
Independent Claim
143 words
An apparatus for measuring cellular properties comprising: (a) a scanner for scanning the cell population with a beam to produce digital data samples, different said samples representing different locations within the cell population; (b) a memory for storing said digital data; (c) means for locating digital data samples in memory corresponding to a cell within said population; (d) means for defining a neighborhood around the located cell; (e) an adder for summing all samples of optical data in a neighborhood to generate an optical neighborhood value for that neighborhood; (f) a comparator for comparing each sample in a neighborhood with a preselected morphology threshold to generate a binary neighborhood pattern of samples either above or below said threshold; and (g) means for generating optical properties corresponding to said optical neighborhood value and morphological properties corresponding to said binary neighborhood pattern for each cell.
Claim 44
Independent Claim
196 words
A method for generating calibrated optical data that characterizes a population of cells in which a number of the cells has a constant optical value, the method comprising: (a) scanning the cell population with a beam along a scan line to produce digital data samples, different said samples representing different locations along said scan line within the cell population; (b) storing said digital data; (c) locating a cell within said population; (d) defining a neighborhood around the located cell, said neighborhood containing optical data derived from said stored digital data samples within said neighborhood; (e) determining the maximum digital data sample within said neighborhood and recentering the neighborhood with respect to said maximum digital data sample; (f) summing all optical data values in a recentered neighborhood to generate an optical neighborhood value for that neighborhood; (g) processing said optical data representing individual cells to generate an illumination normalization factor to correct optical data for illumination differences as a function of cell location along the scan line; and (h) correcting optical data for each cell at different locations along the scan line by multiplying the optical neighborhood value for that cell by the corresponding illumination normalization factor.
Claim 45
Independent Claim
196 words
A method for generating calibrated optical data that characterizes a population of cells in which a number of the cells has a constant optical value, the method comprising: (a) scanning the cell population with a beam along a scan line to produce digital data samples, different said samples representing different locations along said scan line within the cell population; (b) storing said digital data; (c) locating a cell within said population; (d) defining a neighborhood around the located cell, said neighborhood containing optical data derived from said stored digital data samples within said neighborhood; (e) determining the maximum digital data sample within said neighborhood and recentering the neighborhood with respect to said maximum digital data sample; (f) summing all optical data values in a recentered neighborhood to generate an optical neighborhood value for that neighborhood; (g) processing said optical data representing individual cells to generate an illumination normalization factor to correct optical data for illumination differences as a function of cell location along the scan line; and (h) correcting optical data for each cell at different locations along the scan line by multiplying the optical neighborhood value for that cell by the corresponding illumination normalization factor.
Claim 46
Dependent Claim
106 words
The method of claim 35, wherein said processing of said optical data comprises: (a) determining the most frequently occurring optical neighborhood value to define a mode optical value; (b) selecting a subpopulation of cells with optical neighborhood values within a predetermined range around said mode optical value; (c) determining a set of average optical neighborhood values of cells in the subpopulation as a function of cell location along the scan line; and (d) computing an array of correction coefficients equal to the ratio of the mode optical value to the average optical value for each location along the scan line to generate an illumination normalization factor.
Claim 47
Dependent Claim
32 words
The method of claim 36, wherein said predetermined range of optical neighborhood values selecting said subpopulation of cells is from plus or minus 2 to 6 percent of said mode optical value.
Claim 48
Dependent Claim
17 words
The method of claim 36, wherein said subpopulation of cells comprises cells in the resting phase (G₀).
Claim 49
Dependent Claim
17 words
The method of claim 36, wherein said subpopulation of cells comprises cells of substantially the same size.
Claim 50
Dependent Claim
90 words
The method of claim 36, wherein the steps of computing an array of correction coefficients equal to the ratio of the mode optical value to the average optical value for each location along the scan line to generate an illumination normalization factor, and correcting optical data for each cell at different locations along the scan line by multiplying the optical neighborhood value for that cell by the corresponding illumination normalization factor, are repeated iteratively for initially calibrated optical data to compute new illumination normalization factors until said factors are stabilized.
Claim 51
Independent Claim
242 words
A method for generating calibrated optical data that characterizes a population of cells, the method comprising: (a) scanning a population of calibration particles comprising a known constant parameter with a beam along a scan line to produce digital calibration data samples, different said calibration samples representing different locations along said scan line; (b) storing said digital calibration data; (c) locating a calibration particle within said population; (d) defining a neighborhood around the located particle, said neighborhood containing optical calibration data derived from said stored digital calibration data samples within said neighborhood; (e) determining the location of a maximum digital data calibration sample within said neighborhood and recentering the neighborhood with respect to said maximum digital calibration data sample; (f) summing all optical calibration data values in a recentered neighborhood to generate an optical neighborhood calibration value for that neighborhood; (g) processing said optical calibration data to generate an illumination normalization factor to correct optical data for illumination differences as a function of particle location along the scan line; (h) scanning the cell population with a beam along said scan line to produce digital data samples, different said samples representing different locations along said scan line within the cell population; (i) storing said digital data; (j) deriving optical data from said stored digital data; and (k) correcting optical data for each cell at different locations along the scan line by multiplying the optical data for that cell by the corresponding illumination normalization factor.
Claim 52
Independent Claim
242 words
A method for generating calibrated optical data that characterizes a population of cells, the method comprising: (a) scanning a population of calibration particles comprising a known constant parameter with a beam along a scan line to produce digital calibration data samples, different said calibration samples representing different locations along said scan line; (b) storing said digital calibration data; (c) locating a calibration particle within said population; (d) defining a neighborhood around the located particle, said neighborhood containing optical calibration data derived from said stored digital calibration data samples within said neighborhood; (e) determining the location of a maximum digital data calibration sample within said neighborhood and recentering the neighborhood with respect to said maximum digital calibration data sample; (f) summing all optical calibration data values in a recentered neighborhood to generate an optical neighborhood calibration value for that neighborhood; (g) processing said optical calibration data to generate an illumination normalization factor to correct optical data for illumination differences as a function of particle location along the scan line; (h) scanning the cell population with a beam along said scan line to produce digital data samples, different said samples representing different locations along said scan line within the cell population; (i) storing said digital data; (j) deriving optical data from said stored digital data; and (k) correcting optical data for each cell at different locations along the scan line by multiplying the optical data for that cell by the corresponding illumination normalization factor.
Claim 53
Dependent Claim
90 words
The method of claim 41, wherein said processing of said optical calibration data comprises: (a) determining the most frequently occurring optical calibration neighborhood value to define a mode optical calibration value; (b) determining a set of average optical calibration neighborhood values of particles as a function of particle location along the scan line; and (c) computing an array of correction coefficients equal to the ratio of the mode optical calibration value to the average optical calibration value for each location along the scan line to generate an illumination normalization factor.
Claim 54
Independent Claim
25 words
The method of any one of claims 1, 14, 17, 22, 35, or 41, wherein the location of each cell found is recorded and stored.
Claim 55
Independent Claim
45 words
The method of any one of claims 1, 14, 17, 22, 35, or 41, wherein the time corresponding approximately to the time the digital data was acquired is recorded and stored as synchronous digital time data, whereby each sample has a corresponding digital time point.
Claim 56
Independent Claim
29 words
The method of any one of claims 1, 14, 17, 22, or 35, wherein said cell locating step comprises comparing 8 said stored digital data to a preselected threshold.
References Cited (Backward Citations)
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